LOCUS       sequence-314593-        3129 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Acceptor for golden gate assembly with BsaI overhangs GCTT-CCAT. 
            Generates plasmids for SP6-driven TNT SP6 Coupled Wheat Germ Extract
            System .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3129)
  AUTHORS   Dudley QM, Cai YM, Kallam K, Debreyne H, Carrasco Lopez JA, Patron 
            NJ
  TITLE     Biofoundry-assisted expression and characterization of plant 
            proteins.
  JOURNAL   Synth Biol (Oxf). 2021 Sep 11;6(1):ysab029. doi: 
            10.1093/synbio/ysab029. eCollection 2021.
  PUBMED    34693026
REFERENCE   2  (bases 1 to 3129)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Synth Biol 
            (Oxf)."; date: "2021-09-11"; volume: "6(1):ysab029. doi"; pages: " 
            10.1093/synbio/ysab029. eCollection 2021"
COMMENT     Sequence Label: pEPQD0KN0285
FEATURES             Location/Qualifiers
     source          1..3129
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     52..71
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      182..770
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     671..690
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        889..907
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     889..906
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(1100..1423)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     1105..1124
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1318..1340
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1332..1349
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1333..1349
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1350..1406
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1419..1435)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1443..1459
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1467..1473,1474..1491,1492..1497))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1512..1533
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2255..2271)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2295..2313)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2309..2356
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(join(2374..3129,1..60))
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3097..3116
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 atccatgttg gaatttaatc gcggcttcga gcaagacgtt tcccgttgaa tatggctcat
       61 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg atgatatatt
      121 tttatcttgt gcaatgtaac atcagagatt ttgagacaca acgtgagatc aaaggatctt
      181 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
      241 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
      301 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
      361 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
      421 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
      481 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
      541 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
      601 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
      661 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
      721 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
      781 acgcgatccc gcgacgaata agccagatgc tacacaatta ggcttgtaca tactgtcgtt
      841 agaacgcggc tacaattaat acataacctt atgtatcata cacatacgat ttaggtgaca
      901 ctatagaact cacctatctc cccaacacct aataacattc aatcactctt tccactaacc
      961 acctatctac atcaccaaga tatcactagt tctcgagctc catagagacc gtcacagctt
     1021 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     1081 ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
     1141 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc
     1201 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     1261 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     1321 agtcacgacg ttgtaaaacg acggccagtg aattcgagct cggtacccgg ggatcctcta
     1381 gagtcgacct gcaggcatgc aagcttggcg taatcatggt catagctgtt tcctgtgtga
     1441 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     1501 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     1561 cagtcgggaa acctgtcgtg ccagctaggt ctcagcttcg ggttataatt acctcaggtc
     1621 gacgtcccat ggttttgtat agaatttacg gctagcgccg gatgcgacgc cggtcgcgtc
     1681 ttatccggcc ttcctatatc aggcggtgtt taagacgccg ccgcttcgcc caaatcctta
     1741 tgccggttcg acgactggac aaaatactgt ttatcttccc agcgcaggca ggttaatgta
     1801 ccaccccagc agcagccggt atccagcgcg tatatacctt ccggcgtacc tttgccctcc
     1861 agcgatgccc agtgaccaaa ggcgatgctg tattcttcag cgacagggcc aggaatcgca
     1921 aaccacggtt tcagtggggc aggggcctct tccggcgatt cttactagct agtatgcata
     1981 ggtgctgaaa tataaagttt gtgtttctaa aacacacgtg gtacgtacga taacgtacag
     2041 tgtttttccc tccacttaaa tcgaagggta gtgtcttgga gcgcgcggag taaacatata
     2101 tggttcatat atgtccgtag gcacgtaaaa aaagcgaggg attcgaattc ccccggaacc
     2161 cccggttggg gcccacgcct cgatcgagca aaaaaaaaaa aaaagaaaaa aaaaaaaaaa
     2221 aaagctttcc cgcggccagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     2281 gttatccgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     2341 cttgaggggt tttttgctga aagccaattc tgattagaaa aactcatcga gcatcaaatg
     2401 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
     2461 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
     2521 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
     2581 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagctt
     2641 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
     2701 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc
     2761 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
     2821 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
     2881 cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
     2941 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
     3001 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     3061 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     3121 taaatcagc
//
